Human monocyte apoptosis is specifically induced by gel-purified mycobacterial RNA.

<p><i>A.</i> SDS-PAGE with ethidium bromide (left) and silver stained (right) gel-purified RNA (gpRNA) from F7 and treated with RNaseV1 (gpRNA+RNaseV1). <i>B.</i> Monocyte apoptosis induced by gpRNA untreated or treated with RNaseV1, anti-CD95 untreated or treated with RNaseV1 (anti-CD95+RNaseV1) and rabbit mRNA. Significance as described above.</p

The <i>M. tuberculosis</i> RNA's activity is caspase-8 dependent.

<p><i>A.</i> Flow cytometry demonstrated <i>M. tuberculosis</i> H37Rv RNA (gpRNA) activated caspase-8. Human monocytes were stimulated with 1 µg/ml of purified <i>M. tuberculosis</i> H37Rv RNA gpRNA (open histogram with dotted line) or PPD (open histograms with solid line) for 48 h and caspase activity was determined by flow cytometry after staining with FLICA-YVAD-FMK (caspase-1), FLICA-DEVD-FMK (caspase-3), or FLICA-LETD-FMK (caspase-8). Tissue culture medium without RNA or PPD was used as the control (closed histogram). <i>B.</i> Stimulation of apoptosis based on annexinV staining was measured for monocytes stimulated with gpRNA or PPD (top histogram) and when the monocytes were pretreated for 1 h with 10 nM caspase-1 (YVAD-FMK), caspase-3 (DEVD-FMK), or caspase-8 (LETD-FMK) inhibitors (lower histograms).</p

RNA in DEAE-Sepharose Fraction 7 induces apoptosis in human monocytes.

<p><i>A.</i> Apoptosis induced by DEAE-Sepharose Fraction 7 (F7), F7 treated with proteinase K (F7+ProtK), F7 treated with DNase1 (F7+DNase1), F7 treated with RNaseV1 (F7+RNaseV1), CF, CF treated with RNaseV1 (CF+RNaseV1), CF-Man, CF-Man treated with RNaseV1 (CF-Man+RNaseV1). Apoptosis was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive. *** significance p<0.001, ** significance p<0.01 as compared to unstimulated control or between treated samples. <i>B.</i> SDS-PAGE with ethidum bromide staining and <i>C.</i> silver staining of F7 and F7 treated with proteinase K, DNase1, or RNaseV1.</p

Separation of CF by ConA affinity chromatography and DEAE-Sepharose chromatography yielded a fraction with potent apoptotic activity in human monocytes.

<p><i>A.</i> Apoptosis induced by DEAE-Sepharose fractions 1 to 7 (F1 to F7) and unfractionated CF was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive *** significance p<0.001, ** significance p<0.01, * significance p<0.05 as compared to unstimulated control. <i>B.</i> SDS-PAGE and silver staining of fractions of CF after ConA affinity (CF-Man) and DEAE-Sepharose chromatography (F1 to F7). Mw denotes the molecular mass standards.</p

Sequence analysis of cloned extracellular mycobacterial RNA fragments present in <i>M. tuberculosis</i> CF.

1<p>The specific regions of the RNA sequences represented by each clone are depicted in Figure S1.</p>2<p>The number of clones represents the total number of sequences observed for each RNA species.</p

<i>M. tuberculosis</i> H37Rv RNA altered human monocyte's ability to control <i>M. tuberculosis</i> infection.

<p>CFUs were determined for human monocytes infected with <i>M. tuberculosis</i> H37Rv and incubated for four days in the presence of 1 µg/ml of <i>M. tuberculosis</i> H37Rv CF, purified RNA (gpRNA), or purified RNA digested with RNaseV1 (gpRNA+RNaseV1). The presence of CF or gpRNA resulted in a significant increase in CFUs as compared to the untreated infected monocytes (Control). Data represent the mean ± SEM of 3 replicates of the same experiment (*p<0.03).</p

ROC curves showing the predictive capacity of the level of mitochondrial damage to predict active TB development in household contacts of smear-positive patients with or without BCG vaccination.

<p>The gray area shows the confidence interval of AUC (Area Under Curve),Se (Sensitivity), Sp (Specificity).</p

Effect of PPD stimulation on mitochondrial and cell membrane damage in mononuclear phagocytes from household contacts of smear-positive patients in Colombia.

<p>The level of mitochondrial damage was calculated as the difference between the percentage of PPD stimulated cells minus the percentage of non-stimulated cells for mitochondrial damage and cell membrane damage.</p

Distribution of the percentage of monocytes with mitochondrial damage and membrane damage in peripheral blood mononuclear cells (PBMC) cultures from household contacts stimulated and non-stimulated with PPD.

<p>The inset shows the Median (Me) of the percentage of monocytes with mitochondrial (Panels A and C) and cell membrane damage (Panels B and D).Stimulated and non-stimulated cultures were compared using the Wilcoxon test.</p

Incidence of tuberculosis according to the level of mitochondrial damage in household contacts of smear-positive patients with or without BCG vaccination.

<p>The level of mitochondrial damage was categorized by quartiles. The trend in decreasing incidence of active TB was assessed with chi square test for trend (chi square test for trend = 8.66, p = 0.003).</p